The quantification and identification of analytes present in samples derived from a subject is important in the medical arena. In particular, the quantification and identification of analytes from a sample can greatly assist in the diagnoses and treatment of numerous diseases.
For example, nucleic acid analysis is a widely used technique in medical diagnostics. Current techniques of nucleic acid analysis routinely quantify several hundred molecular copies of target nucleic acids per milliliter of patient sample. This requires substantial molecular multiplication, often through a polymerase chain reaction (PCR), to achieve sufficiently high nucleic acid concentrations for detection with conventional laboratory equipment. This molecular amplification requires nucleic acid purification prior to multiplication, which is time consuming, expensive, difficult to control, and has limited accuracy. The final assay result is highly dependent on closely controlling several multistep processes. Currently, available viral load assays report log (rather than linear) values of nucleic acid concentration, at least in part due to limited precision and accuracy inherent in current techniques.
The amplification techniques described above require purified nucleic acid samples free of competing or interfering contaminants. Contaminants can be present in the sample and may include enzymes, proteins, hemoglobin, bacteria, particulate, etc. Alternatively, contaminants can come from the purification system used and may include organic solvents, salts, metal ions, etc. Removing contaminants that interfere with nucleic acid amplification (reverse transcription, amplification, etc.) as well as removing contaminating substances that interfere with detection (hybridization, fluorescence, etc.) requires undertaking time-consuming processes.
Techniques exist to purify nucleic acids. For example, multiple organic reagent extraction/precipitation using chloroform, phenol, and lower alcohols have been used for many years to isolate and purify nucleic acids for subsequent analysis. Additional purification techniques include chromatographic techniques using ion-exchange chromatography, reversed phase chromatography, affinity chromatography, and various combinations thereof.
Silica based nucleic acid purification techniques are currently in wide commercial use. These systems purify nucleic acids through reversible binding (precipitation) onto silica and its derivatives in the presence of chaotropic agents and/or organic solvents. Typically, these systems dilute patient samples containing nucleic acids into 5+ molar guanidine thiocyanate (GTC). This mixture reacts at room temperature, then equal volumes of neat ethyl alcohol are added and vortexed. This mixture is exposed to high surface area silica, where the nucleic acids bind almost immediately. The silica is recovered, rinsed several times with solutions containing GTC and ethyl alcohol, dried, and then the bound nucleic acids are eluted with low salt water. Several versions of this technique exist, and are differentiated based on the silica-based binder used. For instance, one system uses a small filtration or spin column containing a thick silica fiber mat for binding. Other systems use glass beads, magnetic silica based beads, silica impregnated filters, etc. These systems add a carrier nucleic acid (co-precipitant) to permit isolation and purification of very low concentrations of target nucleic acid. While silica based nucleic acid purification techniques adequately perform their intended function, these techniques are time-consuming and expensive.
As with purifying nucleic acids, many techniques exist for immobilizing nucleic acids. To facilitate nucleic acid capture, surfaces are often treated with a variety of chemicals that bind to nucleic acids when the surface is contacted with a solution containing the nucleic acid. For example, covalent attachment of organic compounds to glass and aluminum oxide is known. Such attachment may involve the use of silanization reagents to completely modify the native oxide surface for control of charge, hydrophobic effects, etc., as well as to permit covalent attachment of nucleic acids, proteins, polymers, etc. to effect capture of specific target molecules to the surface. Quartz, glass, and silicon substrates with various surface chemistries have been used to capture nucleic acids for optical molecular detection. These techniques require target nucleic acids to diffuse to the reactive surface for immobilization or capture. At low concentrations, these diffusion-controlled reactions require hours to complete. These techniques are time-consuming, expensive, and may not be suitable for rapid optical molecular detection techniques.
Accordingly, it would be desirable to have materials, devices, and methods for the rapid, inexpensive, and efficient isolation of an analyte from a sample. It is also desirable to remove any contaminants that may interfere with further manipulation (e.g., quantification and identification) of the analyte once it has been removed from the sample.